This disclosure relates to dynamic plasmonics-enabled signal enhancement, devices comprising the same, and methods using the same.
Fluorescence-based and surface enhanced Raman spectroscopy (SERS) are beginning to be widely used in molecular imaging and diagnostics. Techniques using fluorescence include fluorescence lifetime imaging (FLIM), where the imaging of fluorescence lifetime provides a robust means of acquiring spatially resolved information regarding the local environment of a distributed fluorophore in biological tissue and other heterogeneous or turbid media. Other techniques that use picosecond time resolution and nanometer spectral resolution are Foerster's Resonance Energy Transfer (FRET), Fluorescence Activated Cell Sorting (FACS), Fluorescence Lifetime Micro-Spectroscopy (FLMS), polarized fluorescence recovery or redistribution after photobleaching (PFRAP), or the like.
Fluorescence-based techniques generally use a light source for exciting a fluorophore in a biological molecule that is to be imaged or analyzed. The light source can be a lamp source that provides a continuous light output from 250 nanometers (nm) to 750 nm. Sets of optical devices, such as dichroic filters, prisms, or the like, are employed in such a fluorescence-based technique to select the desired excitation and emission wavelengths. Versatility is one of the advantages of such a fluorescence-based technique. The user can change the electromagnetic spectrum by changing the lamp source.
Changing the lamp source has a number of drawbacks. The fluorescence signal emitted by the molecule can be weak depending upon the lamp source used for the irradiation, as a result of which the lamp source may have to be changed to stimulate fluorescence to from an unknown specimen whose molecular imaging or structural features are desired. The sensitivity of instruments containing the lamp source is therefore not always satisfactory.
Another commonly used light source is a laser diode. Laser diodes emit strong monochromatic radiation and therefore promise higher sensitivities. Laser diodes are however, expensive to produce and are not tunable. Having a collection of lasers to probe multiple species of biomolecules with different fluorescent fluorophores is therefore practically difficult. FIG. 1 depicts a conventional fluorescence-based technique using a laser to perform molecular imaging and diagnostics. In FIG. 1, a beam of light from a light source irradiates a molecule whose structure is desired. The molecule produces a fluorescence signal that is then analyzed for the structure of the molecule. However, the fluorescence signal emitted by the molecule can be weak depending upon the light source used for the irradiation.
Poorly performing substrates have plagued surface enhanced Raman spectroscopy (SERS) as an analytical technique since its discovery in 1977 and have effectively limited its acceptance as a reliable method for chemical analysis. Despite the discovery of single molecule sensitivity for SERS in 1997 and the subsequent explosion of interest in SERS, little progress has been made toward the development of useful substrates suitable for commercial manufacturing.
Raman spectroscopy is a chemical analysis method in which monochromatic radiation interacts with molecules (i.e., analyte molecules) and wherein the radiation is shifted in frequency through a process known as scattering. The frequency shift of the scattered radiation is equal to the vibrational frequency of the bonds between atoms in the molecule. Thus, molecules with many bonds produce scattered radiation of many frequencies. Since the vibrational frequencies of most bonds are constant (and are well known), measuring the spectrum of scattered radiation allows the frequency shifts to be determined and the identity of bonds in the molecules to be deduced. The intensity of the scattered radiation is proportional to the number of analyte molecules irradiated, so a Raman spectrum may be used to measure the amount of analyte present and the frequency shifts allow the identification of the analyte. Raman scattering is an extremely inefficient process where only one in 108 incident photons is Raman scattered. To be useful as a sensor, it is desirable for the scattering process to be amplified.
If the scattering process could be amplified, SERS is a vibrational spectroscopic technique that may offer higher sensitivity and versatility
Historically, a number of challenges have existed that have inhibited the successful development and commercialization of SERS substrates. SERS substrates producing enhancement factors of greater than or equal to about 107 for a wide range of analyte molecules do not exist and known substrates show large enhancements for an extremely limited range of highly conjugated organic molecules such as dyes.
Fabrication methods are complex multi-step laboratory processes that are not suitable for scale up to production manufacturing levels. Finally, substrate morphology on the nanoscale is difficult to reproduce and the relationship between substrate nanoscale morphology and SERS enhancement factors is poorly understood.
It is therefore desirable to have an analytical technique that is rapid, inexpensive and that can be tuned to accomplish detection, diagnosis and/or imaging in real time.